Protein A is a cell wall component produced by nearly all strains of Staphylococcus aureus (see e.g. Forsgren, A., Infection and Immunity 2: 672-673 [1970]); and Sjoquist, J. et al., Eur. J. Biochem. 30: 190-194 [1972]). Protein A is useful in that it binds strongly and specifically to the Fc portion of immunoglobulin IgG from a variety of mammalian sources, including human (Kronvall, G. et al., J. Immunol. 103: 828-833 [1969]). Thus this protein has been used in diagnostic applications and has potential therapeutic value.
In most S. aureus strains, at least 70% of the Protein A produced is covalently linked to the peptidoglycan of the cell wall (Sjoquist, J. et al., Eur. J. Biochem. 30: 190-194 [1972]. The site of attachment is the C-terminal region of the Protein A molecule (Sjodahl, J., Eur. J. Biochem. 73: 343-351 [1977]). Some Protein A (15-30%) is generally excreted into the growth medium, and there are several circumstances under which the fraction of Protein A which is excreted can be increased. Some methicillin resistant strains of S. aureus excrete essentially all their Protein A (Lindmark, R. et al., Eur. J. Biochem. 74: 623-628 [1977]). Low levels of puromycin increase the amount of excreted Protein A, presumably by truncating the protein and thereby eliminating its C-terminal cell wall attachment site, and protoplasts excrete nearly all the Protein A which they synthesize (Movitz, J., Eur. J. Biochem. 68: 291-299 [1976]).
Protein sequence information is available for Protein A from S. aureus strain Cowan I (Sjodahl, J., Eur. J. Biochem. 78: 471-490 [1977]). The Cowan I strain contains approximately 2.times.10.sup.5 molecules of Protein A per cell (Sjoquist, J. et al., Eur. J. Biochem. 30: 190-194 [1972]).
Protein A is synthesized in S. aureus only during exponential growth, and synthesis ceases as the culture approaches stationary phase (Movitz, J., Eur. J. Biochem. 48: 131-136 [1974]). The level of synthesis of Protein A in S. aureus is highly variable, and is strongly influenced by the growth conditions in some as yet poorly defined ways (Landwall, P., J. Applied Bact. 44: 151-158 [1978]).
The Protein A gene from S. aureus strain Cowan I has been cloned in E. coli. Lofdahl, S., et al., Proc. Natl. Acad. Sci. USA, 80, 697-701 (1983). This gene is contained in a 2.15 kilobase insert bounded by EcoRV restriction sites. The gene has been inserted into a plasmid and cloned in E. coli, where low levels of expression have been achieved. The chimeric plasmid which contains the Protein A gene has been designated "pSPA1."
Currently, industrial production of Protein A is carried out using mutant strains of S. aureus. A major disadvantage of using S. aureus to produce Protein A is that all available production strains are human pathogens. Although many genetic engineering experiments have been conducted using Escherichia coli, that organism is not suitable for efficient production of Protein A, since it does not export protein outside the cell. Furthermore, E. coli possesses disadvantageous pathogenic properties as well, i.e., produces endotoxins.
There thus remains a need for the production of Protein A by means which are both safe and efficient.